Understanding regulatory mechanisms that maintain pluripotent embryonic cells and control differentiation has fundamental relevance to scientific areas central to human health and disease, including stem cell, developmental, and cancer biology. The novel Geminin (Gem) protein acts together with the SWI/SNF and Polycomb (PcG) chromatin regulatory complexes as key transcriptional regulators of cell lineage specification and differentiation in embryonic and embryonic stem (ES) cells. Dysregulation of Gem, SWI/SNF and PcG activities is also a pivotal aspect of multiple human malignancies. A major focus of my laboratory is to understand how this core transcriptional regulatory switch operates and to define approaches for manipulating it in both normal ES and progenitor cell contexts and in disease. Our preliminary results support a model where Gem maintains pluripotent and/or multipotent progenitor cells by direct transcriptional repression of cell lineage commitment and differentiation regulatory genes. We hypothesize that Gem does this: 1. by directly cooperating with PcG complexes to repress the expression of common target loci. In support of this, we recently defined Gem-repressed target genes, and found these overlapped strikingly with direct targets of PcG repression to block cell lineage commitment. 2. Gem also acts in neuronal progenitor cells to regulate differentiation timing. For this activity, we hypothesize that Gem antagonizes target gene transactivation by the coordinated activities of SWI/SNF and Neurogenin2 and NeuroD, neural bHLH transcription factors required for neuronal fate commitment and differentiation. Here, in Aim 1 we will use an innovative, high-throughput approach to identify enhancers that are bound and regulated by Neurogenin2 and NeuroD in their direct target genes, and negatively regulated by Gem. This will fill an existing gap in our knowledge by defining transcriptional regulatory mechanisms and networks through which Neurogenin2 and NeuroD perform essential roles in neurogenesis and will provide a necessary sequence context for analyzing Gem's role in this regulation. In Aims 2 and 3, we will analyze how Gem, SWI/SNF and PcG are recruited to and mechanistically control expression of Gem target genes and how disrupting functional interplay between these activities perturbs target gene transactivation at the chromatin level during neuronal differentiation. Together, these studies will elucidate mechanisms and logic of a core transcriptional regulatory switch required to maintain embryonic stem and progenitor cells and to regulate differentiation. Information and tools that we develop here will provide a foundation for diagnostic and therapeutic manipulations of this regulatory switch in both normal stem and progenitor cells and in disease contexts such as malignancy. PUBLIC HEALTH RELEVANCE: The proposed work will determine mechanisms of action of a central regulatory switch controlling gene expression in embryonic stem cells and dysregulated in many aggressive, therapy-resistant forms of cancer. Information and tools that we develop here will provide a critical foundation for manipulating this regulatory switch for both diagnostic and therapeutic purposes in normal stem cell contexts and in treating human malignancies.